文献类型：期刊文献

中文题名：HBD的N端融合蛋白的跨膜转导作用

英文题名：Transmembrane Activity of Heparin-Binding Domain Fused at N-Terminal of Heterologous Protein

作者：杜璇[1];顾红艳[2];李亚丹[2];吴珍[2];赵健[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]浙江日升昌药业有限公司,浙江东阳322100

年份：2014

卷号：40

期号：2

起止页码：176

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2013_2014】；

基金：国家自然科学基金(81171449)

语种：中文

中文关键词：穿膜肽;肝素结合域;HeLa细胞;EGFP的N端

外文关键词：cell penetrating peptides; HBD; HeLa cell; N-terminus of EGFP

摘要：研究人源性穿膜肽HBD的N端融合蛋白的跨膜转导作用。通过DNA重组技术将HBD融合在EGFP蛋白的氨基端(N端),构建重组质粒pET28b-HBD-EGFP-Histag,经IPTG诱导表达和Ni 2+-NTA纯化,获得重组蛋白HBD-EGFP。通过激光共聚焦显微镜定性观察及流式细胞仪定量检测发现,融合在EGFP氨基端的HBD跨膜转导效率比融合在EGFP羧基端(C端)的HBD转导效率提高了约10倍。实验结果表明,N端融合的HBD-EGFP对于外源蛋白具有跨膜运输效果,且比C端融合的HBD具有更高的跨膜转导效率。该结果可为HBD在抗癌药物递送方面的有效应用提供理论依据。
To study the transmembrane activity of Heparin-Binding Domain（HBD）derived from human fused at N-terminal of heterologous protein,the expression plasmid pET28b-HBD-EGFP-Histag was constructed by genetically fusing HBD to the N-terminal of enhanced green fluorescent protein（EGFP）.The fusion protein HBD-EGFP was expressed in E.coli and purified by Ni 2＋-NTA affinity chromatography.The results show that green fluorescence can be observed in HeLa cells using laser scanning confocal microscope when HeLa cells were co-cultured with HBD-EGFP.Flow-cytometry results show that the transmembrane efficiency of HBD fused at the N-terminal of EGFP is improved about 10 times compared with the HBD fused at the C-terminal of EGFP.These results demonstrated that HBD fused to N-terminal of heteologous protein exhibited tansmembrane activity.The HBD fused at the Nterminal of EGFP had a higher efficiency than the HBD fused at the C-terminal of EGFP.These results will provide theoretical foundation for the efficient application of HBD in the field of the delivery of anticancer drugs.